Regulation Of Cytokinesis Research Articles

Evidence for biomolecular condensates of MatP in spatiotemporal regulation of the bacterial cell division cycle.

An increasing number of proteins involved in bacterial cell cycle events have been recently shown to undergo phase separation. The resulting biomolecular condensates play an important role in cell cycle protein function and may be involved in development of persister cells tolerant to antibiotics. Here we report that the E. coli chromosomal Ter macrodomain organizer MatP, a division site selection protein implicated in the coordination of chromosome segregation with cell division, forms biomolecular condensates in cytomimetic systems. These condensates are favored by crowding and preferentially localize at the membrane of microfluidics droplets, a behavior probably mediated by MatP-lipid binding. Condensates are negatively regulated and partially dislodged from the membrane by DNA sequences recognized by MatP ( matS ), which partition into them. Unexpectedly, MatP condensation is enhanced by FtsZ, a core component of the division machinery previously described to undergo phase separation. Our biophysical analyses uncover a direct interaction between the two proteins, disrupted by matS sequences. This binding might have implications for FtsZ ring positioning at mid-cell by the Ter linkage, which comprises MatP and two other proteins that bridge the canonical MatP/FtsZ interaction. FtsZ/MatP condensates interconvert with bundles in response to GTP addition, providing additional levels of regulation. Consistent with discrete foci reported in cells, MatP biomolecular condensates may facilitate MatP's role in chromosome organization and spatiotemporal regulation of cytokinesis and DNA segregation. Moreover, sequestration of MatP in these membraneless compartments, with or without FtsZ, could promote cell entry into dormant states that are able to survive antibiotic treatments.

Characterization of Ksg1 protein kinase-dependent phosphoproteome in the fission yeast S. pombe

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

NudCL2 is required for cytokinesis by stabilizing RCC2 with Hsp90 at the midbody.

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells. Midbody, a protein-dense organelle that forms at the intercellular bridge, is indispensable for successful cytokinesis. However, the regulatory mechanism of cytokinesis at the midbody still remains elusive. Here, we unveil a critical role for NudC-like protein 2 (NudCL2), a co-chaperone of heat shock protein 90 (Hsp90), in cytokinesis regulation by stabilizing regulator of chromosome condensation 2 (RCC2) at the midbody in mammalian cells. NudCL2 localizes at the midbody, and its downregulation results in cytokinesis failure, multinucleation, and midbody disorganization. Using iTRAQ-based quantitative proteomic analysis, we find that RCC2 levels are decreased in NudCL2 knockout (KO) cells. Moreover, Hsp90 forms a complex with NudCL2 to stabilize RCC2, which is essential for cytokinesis. RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells. Importantly, ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion, but not vice versa. Together, our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody.

Identification of differentially expressed genes and screening for key genes involved in ovarian cancer prognosis: An integrated bioinformatics and network analysis approach

Objectives: Ovaries are important and essential organs of animals in producing and releasing eggs. Ovarian cancer (OvCa) is one of the most prevalent lethal gynecological malignancies with a lack of distinct biomarkers. Advances in high-throughput genomic data and the continued refinement of bioinformatics tools enable the identification of potential biomarkers. Leveraging these insights, we can employ systems biology approaches to enhance the accuracy of diagnosis and prognosis. Material and Methods: A comparative analysis was conducted between normal and tumor samples, employing bioinformatics software and tools. Differential expression analysis utilized fold-change statistics, while DAVID 6.8 software was used to perform gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The protein-protein interaction (PPI) network was constructed differentially expressed genes (DEGs) using Search Tool for the Retrieval of Interacting Genes database, and Cytoscape 3.9.1, along with its Molecular Complex Detection and CytoHubba plugins, facilitated network visualization, analysis, and module detection. Hub gene expression and overall survival were explored through the Kaplan–Meier plotter, while Gene Expression Profiling Interactive Analysis 2 analyzed the tumor stage of OvCa patients. Hub genes protein expression was analyzed using the human protein atlas database through immunostaining results. The NetworkAnalyst program and Cytoscape were employed to analyze and visualize the transcription factor-hub gene associations. Subsequently, single-nucleotide variation, methylation, and pathway activity of hub genes were examined. Validation of hub genes messenger RNA expression was done using quantitative real-time polymerase chain reaction analysis. Results: 607 DEGs, including 248 upregulated and 359 downregulated genes, were identified. The top 20 candidate genes were screened out through PPI network analysis. We discovered that the genes BUB1 Mitotic Checkpoint Serine/Threonine Kinase B (BUB1B), Cyclin A2 (CCNA2), Mitotic Arrest Deficient 2 Like 1 (MAD2L1), Protein Regulator of Cytokinesis 1 (PRC1), Thyroid Hormone Receptor Interactor 13 (TRIP13), and ZW10 Interacting Kinetochore Protein (ZWINT) exhibited significant importance in OvCa prognosis. Conclusion: Six genes, BUB1B, CCNA2, MAD2L1, PRC1, TRIP13, and ZWINT (identified as functional hub genes), are probably playing tumor-promotive roles, except TRIP13. All genes product is functionally related to the cell cycle. These can be targeted in quest of potential therapeutics for OvCa treatment.

The Complex Regulation of Cytokinesis upon Abscission Checkpoint Activation.

Cytokinetic abscission is a crucial process that guides the separation of daughter cells at the end of each cell division. This process involves the cleavage of the intercellular bridge, which connects the newly formed daughter cells. Over the years, researchers have identified several cellular contributors and intracellular processes that influence the spatial and temporal distribution of the cytoskeleton during cytokinetic abscission. This review presents the most important scientific discoveries that allow activation of the abscission checkpoint, ensuring a smooth and successful separation of a single cell into two cells during cell division. Here, we describe different factors, such as abscission checkpoint, ICB tension, nuclear pore defects, DNA replication stress, chromosomal stability, and midbody proteins, which play a role in the regulation and correct timing of cytokinetic abscission. Furthermore, we explore the downsides associated with the dysregulation of abscission, including its negative impact on cells and the potential to induce tumor formation in humans. Finally, we propose a novel factor for improving cancer therapy and give future perspectives in this research field.

Transcriptome analysis reveals hepatic disordered lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses due to maternal undernutrition during late pregnancy

Although lipid metabolism in fetal livers under intrauterine growth restriction (IUGR) conditions has been widely studied, the implications of maternal undernutrition on fetal hepatic lipid metabolism, lipotoxic injury, and abnormal development remain largely unknown. Therefore, this study investigated the effects of maternal undernutrition on disordered hepatic lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses using transcriptome analysis. Seventeen singleton ewes were randomly divided into three groups on day 90 of pregnancy: a control group (CG; 0.63 MJ metabolic energy/body weight (ME/BW)0.75/day, n = 5), maternal undernutrition group 1 (MU1; 0.33 MJ ME/BW0.75/day, n = 6), and maternal undernutrition group 2 (MU2; 0.20 MJ ME/BW0.75/day, n = 6). The fetuses were euthanized and recovered on day 130 of pregnancy. The levels of free fatty acids (FFA) in maternal blood (P < 0.01), fetal blood (P < 0.01), and fetal livers (P < 0.05) were increased in the MU1 and MU2 groups, but fetal hepatic triglyceride (TG) levels in the MU2 group (P < 0.01) and β-hydroxybutyrate levels in the MU1 and MU2 groups (P < 0.01) were decreased compared to the CG. Severe inflammatory cell infiltration and increased non-alcoholic fatty liver disease activity scores were observed in MU1 and MU2 fetuses (P < 0.01). Progressive deposition of fetal hepatic reticular fibers and collagen fibers in the fetal livers of the MU1 and MU2 groups and significant hepatic fibrosis were observed in the MU2 fetuses (P < 0.05). Gene set enrichment analysis showed that genes involved in lipid accumulation and FFA beta oxidation were downregulated in both MU groups compared to those in the controls. The fetal liver mRNA expression of the β-oxidation regulator, acetyl-CoA acetyltransferase 1, and the TCA regulator, isocitrate dehydrogenase were reduced in MU1 (P < 0.05) and MU2 (P < 0.01) fetuses, and downregulated mRNA expression of long chain fatty acid CoA ligase 1 (P < 0.05) and glycerol-3-phosphate acyltransferase (P < 0.01) was observed in MU2 fetuses. Differentially expressed genes (DEGs) in MU1 versus CG (360 DEGs) and MU2 versus CG (746 DEGs) were identified using RNA sequencing. Bioinformatics analyses of the 231 intersecting DEGs between MU1 versus CG and MU2 versus CG indicated that neutrophil extracellular traps (NETs) were induced and played a central role in fetal hepatic injury in IUGR sheep. Increased maternal blood myeloperoxidase (MPO) levels (P < 0.01), NE (Elane)-positive areas in fetal liver sections (P < 0.05), and fetal liver MPO protein expression (P < 0.01) were found in the MU1 and MU2 groups; however, MPO levels were reduced in the fetal membrane (P < 0.01) and fetal blood (P < 0.05) in the MU1 group, and in the maternal-fetal placenta and fetal blood in the MU2 group (P < 0.01). Analysis of gene expression trends in the intersecting DEGs between MU1 versus CG (129 DEGs) and MU2 versus CG (515 DEGs) further revealed that 30 hub genes were essential regulators of the G2/M cell cycle, all of which were associated with hepatocellular carcinoma. G0/G1 phase cells of the fetal liver were reduced in the MU1 (P < 0.05) and MU2 (P < 0.01) groups, whereas G2/M phase cells were elevated in the MU1 and MU2 groups (P < 0.01). The representatives of upregulated hub genes and fetal liver protein expression of maternal embryonic leucine zipper kinase and protein regulator of cytokinesis 1 were progressively enhanced in the MU1 and MU2 groups (P < 0.01), and topoisomerase II alpha protein expression in the MU2 group (P < 0.05), as expected. These results indicate that FFA overload, severe lipotoxic injury, and NETs were induced, and disease-promoting regulators of the G2/M cell cycle were upregulated in the fetal liver of IUGR sheep. These findings provide new insights into the pathogenesis of impaired hepatic lipid metabolism and abnormal development and the molecular origin of post-natal liver disease in IUGR due to maternal undernutrition. This information can support the development of new therapeutic strategies.

The Activity of YCA1 Metacaspase Is Regulated by Reactive Sulfane Sulfur via Persulfidation in Saccharomyces cerevisiae.

YCA1, the only metacaspase in Saccharomyces cerevisiae, plays important roles in the regulation of chronological lifespan, apoptosis, and cytokinesis. YCA1 has protein hydrolase activity and functions by cleaving itself and target proteins. However, there are few reports about the regulation of YCA1 activity. In this study, we observed that reactive sulfane sulfur (RSS) can inhibit the activity of YCA1. In vitro experiments demonstrated that RSS reacted with the Cys276 of YCA1, the residue central to its protein hydrolase activity, to form a persulfidation modification (protein-SSH). This modification inhibited both its self-cleavage and the cleavage of its substrate protein, BIR1. To investigate further, we constructed a low-endogenous-RSS mutant of S. cerevisiae, BY4742 Δcys3, in which the RSS-producing enzyme cystathionine-γ-lyase (CYS3) was knocked out. The activity of YCA1 was significantly increased by the deletion of CYS3. Moreover, increased YCA1 activity led to reduced chronological lifespan (CLS) and CLS-driven apoptosis. This study unveils the first endogenous factor that regulates YCA1 activity, introduces a novel mechanism of how yeast cells regulate chronological lifespan, and broadens our understanding of the multifaceted roles played by RSS.

Germ fate determinants protect germ precursor cell division by reducing septin and anillin levels at the cell division plane.

Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formincyk-1(ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septinUNC-59 and anillinANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.

PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

Identification of naphthalimide-derivatives as novel PBD-targeted polo-like kinase 1 inhibitors with efficacy in drug-resistant lung cancer cells

Targeting polo-box domain (PBD) small molecule for polo-like kinase 1 (PLK1) inhibition is a viable alternative to target kinase domain (KD), which could avoid pan-selectivity and dose-limiting toxicity of ATP-competitive inhibitors. However, their efficacy in these settings is still low and inaccessible to clinical requirement. Herein, we utilized a structure-based high-throughput virtual screen to find novel chemical scaffold capable of inhibiting PLK1 via targeting PBD and identified an initial hit molecule compound 1a. Based on the lead compound 1a, a structural optimization approach was carried out and several series of derivatives with naphthalimide structural motif were synthesized. Compound 4Bb was identified as a new potent PLK1 inhibitor with a KD value of 0.29 μM. 4Bb could target PLK1 PBD to inhibit PLK1 activity and subsequently suppress the interaction of PLK1 with protein regulator of cytokinesis 1 (PRC1), finally leading to mitotic catastrophe in drug-resistant lung cancer cells. Furthermore, 4Bb could undergo nucleophilic substitution with the thiol group of glutathione (GSH) to disturb the redox homeostasis through exhausting GSH. By regulating cell cycle machinery and increasing cellular oxidative stress, 4Bb exhibited potent cytotoxicity to multiple cancer cells and drug-resistant cancer cells. Subcutaneous and oral administration of 4Bb could effectively inhibit the growth of drug-resistant tumors in vivo, doubling the survival time of tumor bearing mice without side effects in normal tissues. Thus, our study offers an orally-available, structurally-novel PLK1 inhibitor for drug-resistant lung cancer therapy.

Antiparallel microtubule bundling supports KIF15-driven mitotic spindle assembly.

The spindle is a bipolar microtubule-based machine that is crucial for accurate chromosome segregation. Spindle bipolarity is generated by Eg5 (a kinesin-5), a conserved motor that drives spindle assembly by localizing to and sliding apart antiparallel microtubules. In the presence of Eg5 inhibitors (K5Is), KIF15 (a kinesin-12) can promote spindle assembly, resulting in K5I-resistant cells (KIRCs). However, KIF15 is a less potent motor than Eg5, suggesting that other factors may contribute to spindle formation in KIRCs. Protein Regulator of Cytokinesis 1 (PRC1) preferentially bundles antiparallel microtubules, and we previously showed that PRC1 promotes KIF15-microtubule binding, leading us to hypothesize that PRC1 may enhance KIF15 activity in KIRCs. Here, we demonstrate that: 1) loss of PRC1 in KIRCs decreases spindle bipolarity, 2) overexpression of PRC1 increases spindle formation efficiency in KIRCs, 3) overexpression of PRC1 protects K5I naïve cells against the K5I S-trityl-L-cysteine (STLC), and 4) PRC1 overexpression promotes the establishment of K5I resistance. These effects are not fully reproduced by a TPX2, a microtubule bundler with no known preference for microtubule orientation. These results suggest a model wherein PRC1-mediated bundling of microtubules creates a more favorable microtubule architecture for KIF15-driven mitotic spindle assembly in the context of Eg5 inhibition.

Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

KIFC3 is a member of Kinesin-14 family motor proteins, which play a variety of roles such as centrosome cohesion, cytokinesis, vesicles transportation and cell proliferation in mitosis. Here, we investigated the functional roles of KIFC3 in meiosis. Our findings demonstrated that KIFC3 exhibited expression and localization at centromeres during metaphase I, followed by translocation to the midbody at telophase I throughout mouse oocyte meiosis. Disruption of KIFC3 activity resulted in defective polar body extrusion. We observed aberrant meiotic spindles and misaligned chromosomes, accompanied by the loss of kinetochore-microtubule attachment, which might be due to the failed recruitment of BubR1/Bub3. Coimmunoprecipitation data revealed that KIFC3 plays a crucial role in maintaining the acetylated tubulin level mediated by Sirt2, thereby influencing microtubule stability. Additionally, our findings demonstrated an interaction between KIFC3 and PRC1 in regulating midbody formation during telophase I, which is involved in cytokinesis regulation. Collectively, these results underscore the essential contribution of KIFC3 to spindle assembly and cytokinesis during mouse oocyte meiosis.

Abstract 674: Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway

Abstract Aberrant migration and proliferation of cancer cells are the hallmark of metastatic tumors. Protein Regulator of Cytokinesis 1 (PRC1) is a scaffold protein that regulates metabolic pathways of cancer cell proliferation, survival, and metastasis. BKT300 is a small molecule (MW 399.33, C22H25NO6), that selectively inhibits the migration and survival of hematological and solid cancer cells. BKT300 inhibits, at high affinity, both actin and microtubule formation and organization leading to G2/M cell cycle arrest, mitotic catastrophe, and apoptosis through the caspase 3 pathway. These effects are selective to cancer cells and were not observed on normal cells. BKT300 was found to arrest PRC1 in a phosphorylated form at position T481, down regulates the expression of CDC25C and up regulate p21. Suppression of PRC1 using SiRNA ablation inhibited BKT300 induced apoptosis on several tumor cell lines and inhibited the effect of BKT300 on G2M cell cycle arrest. Furthermore, using Biacore SPR, microscale thermophoresis (MST), and ELISA assays we were able to demonstrate the direct binding of BKT300 to PRC1. Beyond its single agent activity, BKT300 was shown to synergize with standard of care (SoC) treatments, including anti PD-1 immunotherapy, BCL-2 inhibitor (Ventoclax), topoisomerase I inhibitor (Irinotecan) topoisomerase II inhibitor (Doxorubicin), anti-metabolite Fluorouracil (5-FU) and alkylating agent (Temozolomide). BKT300 has a remarkable safety profile when administered at high doses to mice rats, minipigs and non-human primates. Treatment with BKT300 showed almost no effects on hematopoiesis and biochemistry in doses far higher than the affective ones. The in-vivo activity of BKT300 was demonstrated in multiple Xenograft, Syngeneic and PDX models in mice. In-vivo treatment with BKT300 resulted in preventive (small tumors) as well as therapeutic (large tumors) activity in triple negative breast cancer (TNBC) xenograft, MDA-MB-453 and MDA-MB-468 models leading to over then 70%-90% reduction in tumor size following one to two treatments cycles. Similar results were obtained using PDX models derived from patients with Colon, Ovarian and Pancreatic cancer. BKT300 is a novel, first in class, targeted anti-cancer treatment that inhibits the activity of the scaffold protein PRC1. BKT300 is expected to provide a new treatment option to, the high unmet medical need of, advanced difficult to treat cancers, specifically ones that overexpress PRC1 and CDC25C. Citation Format: Amnon Peled, Michal Abraham, Hanna Wald, Rakefet Rosenfeld, Orly Eizenberg, Arnon Aharon. Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 674.

Rapid evolution of an adaptive multicellular morphology of Candida auris during systemic infection

Candida auris has become a serious threat to public health. The mechanisms of how this fungal pathogen adapts to the mammalian host are poorly understood. Here we report the rapid evolution of an adaptive C. auris multicellular aggregative morphology in the murine host during systemic infection. C. auris aggregative cells accumulate in the brain and exhibit obvious advantages over the single-celled yeast-form cells during systemic infection. Genetic mutations, specifically de novo point mutations in genes associated with cell division or budding processes, underlie the rapid evolution of this aggregative phenotype. Most mutated C. auris genes are associated with the regulation of cell wall integrity, cytokinesis, cytoskeletal properties, and cellular polarization. Moreover, the multicellular aggregates are notably more recalcitrant to the host antimicrobial peptides LL-37 and PACAP relative to the single-celled yeast-form cells. Overall, to survive in the host, C. auris can rapidly evolve a multicellular aggregative morphology via genetic mutations.

Localized calcium transients in phragmoplast regulate cytokinesis of tobacco BY-2 cells.

Plants exhibit a unique pattern of cytosolic Ca2+ dynamics to correlate with microtubules to regulate cytokinesis, which significantly differs from those observed in animal and yeast cells. Calcium (Ca2+) transients mediated signaling is known to be essential in cytokinesis across eukaryotic cells. However, the detailed spatiotemporal dynamics of Ca2+ during plant cytokinesis remain largely unexplored. In this study, we employed GCaMP5, a genetically encoded Ca2+ sensor, to investigate cytokinetic Ca2+ transients during cytokinesis in Nicotiana tabacum Bright Yellow-2 (BY-2) cells. We validated the effectiveness of GCaMP5 to capture fluctuations in intracellular free Ca2+ in transgenic BY-2 cells. Our results reveal that Ca2+ dynamics during BY-2 cell cytokinesis are distinctly different from those observed in embryonic and yeast cells. It is characterized by an initial significant Ca2+ spike within the phragmoplast region. This spike is followed by a decrease in Ca2+ concentration at the onset of cytokinesis in phragmoplast, which then remains elevated in comparison to the cytosolic Ca2+ until the completion of cell plate formation. At the end of cytokinesis, Ca2+ becomes uniformly distributed in the cytosol. This pattern contrasts with the typical dual waves of Ca2+ spikes observed during cytokinesis in animal embryonic cells and fission yeasts. Furthermore, applications of pharmaceutical inhibitors for either Ca2+ or microtubules revealed a close correlation between Ca2+ transients and microtubule organization in the regulation of cytokinesis. Collectively, our findings highlight the unique dynamics and crucial role of Ca2+ transients during plant cell cytokinesis, and provides new insights into plant cell division mechanisms.

Protein regulator of cytokinesis 1 accentuates cholangiocarcinoma progression via mTORC1-mediated glycolysis.

This study aimed to investigate the expression of protein regulator of cytokinesis 1 (PRC1) in cholangiocarcinoma (CHOL) and elucidate its potential impact as well as the underlying mechanisms governing the progression of CHOL. In this study, we used CHOL cells (HUCCT1, RBE, and CCLP1) and conducted a series of experiments, including qRT-PCR, cell counting kit-8 assays, EdU assays, flow cytometry, wound healing assays, Transwell assays, western blotting, double luciferase assays, and ELISA. Subsequently, a mouse model was established using cancer cell injections. Haematoxylin-eosin staining, along with Ki67 and TUNEL assays, were employed to assess tissue histopathology, cell proliferation, and apoptosis. Our findings revealed significantly elevated PRC1 expression in CHOL. According to bioinformatics analysis, it was found that the increased PRC1 level is correlated with the high tumour grades, metastases, and unfavourable prognoses. Notably, PRC1 knockdown inhibited cell viability, proliferation, migration, and invasion while promoting apoptosis in CHOL cells. Analysing TCGA-CHOL data and utilising transcription factor prediction tools (hTFtarget and HumanTFDB), we identified that genes positively correlated with PRC1 in TCGA-CHOL intersect with predicted transcription factors, revealing the activation of PRC1 by forkhead box protein M1 (FOXM1). Moreover, PRC1 was found to exert regulatory control over glycolysis and the mammalian target of rapamycin complex 1 (mTORC1) pathway in the context of CHOL based on KEGG and GSEA analysis. Collectively, these results underscore the pivotal role of PRC1 in CHOL progression, wherein it modulates glycolysis and the mTORC1 pathway under the regulatory influence of FOXM1.

Processes Controlling the Contractile Ring during Cytokinesis in Fission Yeast, Including the Role of ESCRT Proteins.

Cytokinesis, as the last stage of the cell division cycle, is a tightly controlled process amongst all eukaryotes, with defective division leading to severe cellular consequences and implicated in serious human diseases and conditions such as cancer. Both mammalian cells and the fission yeast Schizosaccharomyces pombe use binary fission to divide into two equally sized daughter cells. Similar to mammalian cells, in S. pombe, cytokinetic division is driven by the assembly of an actomyosin contractile ring (ACR) at the cell equator between the two cell tips. The ACR is composed of a complex network of membrane scaffold proteins, actin filaments, myosin motors and other cytokinesis regulators. The contraction of the ACR leads to the formation of a cleavage furrow which is severed by the endosomal sorting complex required for transport (ESCRT) proteins, leading to the final cell separation during the last stage of cytokinesis, the abscission. This review describes recent findings defining the two phases of cytokinesis in S. pombe: ACR assembly and constriction, and their coordination with septation. In summary, we provide an overview of the current understanding of the mechanisms regulating ACR-mediated cytokinesis in S. pombe and emphasize a potential role of ESCRT proteins in this process.

A kinesin-13 family kinesin in Trypanosoma brucei regulates cytokinesis and cytoskeleton morphogenesis by promoting microtubule bundling.

The early branching eukaryote Trypanosoma brucei divides uni-directionally along the longitudinal cell axis from the cell anterior toward the cell posterior, and the cleavage furrow ingresses along the cell division plane between the new and the old flagella of a dividing bi-flagellated cell. Regulation of cytokinesis in T. brucei involves actomyosin-independent machineries and trypanosome-specific signaling pathways, but the molecular mechanisms underlying cell division plane positioning remain poorly understood. Here we report a kinesin-13 family protein, KIN13-5, that functions downstream of FPRC in the cytokinesis regulatory pathway and determines cell division plane placement. KIN13-5 localizes to multiple cytoskeletal structures, interacts with FPRC, and depends on FPRC for localization to the site of cytokinesis initiation. Knockdown of KIN13-5 causes loss of microtubule bundling at both ends of the cell division plane, leading to mis-placement of the cleavage furrow and unequal cytokinesis, and at the posterior cell tip, causing the formation of a blunt posterior. In vitro biochemical assays demonstrate that KIN13-5 bundles microtubules, providing mechanistic insights into the role of KIN13-5 in cytokinesis and posterior morphogenesis. Altogether, KIN13-5 promotes microtubule bundle formation to ensure cleavage furrow placement and to maintain posterior cytoskeleton morphology in T. brucei.

Discovery and Quantitative Dissection of Cytokinesis Mechanisms Using Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.

Identification of prognostic biomarkers of invasive ductal carcinoma by an integrated bioinformatics approach

Background: Invasive ductal carcinoma (IDC) is the most common type of breast cancer (BC) worldwide. Nowadays, due to its heterogeneity and high capacity for metastasis, it is necessary to discover novel diagnostic and prognostic biomarkers. Therefore, this study aimed to identify novel candidate prognostic genes for IDC using an integrated bioinformatics approach. Methods: Three expression profile data sets were obtained from GEO (GSE29044, GSE3229, and GSE21422), from which differentially expressed genes (DEGs) were extracted for comparative transcriptome analysis of experimental groups (IDC versus control). Next, STRING was utilized to construct a protein interaction network with the shared DEGs, and MCODE and cytoHubba were used to identify the hub genes, which were then characterized using functional enrichment analysis in DAVID and KEGG. Finally, using the Kaplan-Meier tracer database, we determined the correlation between the expression of hub genes and overall survival in BC. Results: We identified seven hub genes (Kinesin-like protein KIF23 [KIF23], abnormal spindle-like microcephaly [ASPM]-associated protein [ASPMAP], Aurora kinase A [AURKA], Rac GTPase-activating protein 1 [RACGAP1], centromere protein F [CENPF], hyaluronan-mediated motility receptor [HMMR], and protein regulator of cytokinesis 1 [PRC1]), which were abundant in microtubule binding and tubulin binding, pathways linked to fundamental cellular structures including the mitotic spindle, spindle, microtubule, and spindle pole. The role of these genes in the pathophysiology of IDC is not yet well characterized; however, they have been associated with other common types of BC, modulating pathways such as Wnt/β-catenin, the epithelial-to-mesenchymal transition (EMT) process, chromosomal instability (CIN), PI3K/AKT/mTOR, and BRCA1 and BRCA2, playing an important role in its progression and being associated with a poor prognosis, thus representing a way to improve our understanding of the process of tumorigenesis and the underlying molecular events of IDC. Conclusions: Genes identified may lead to the discovery of new prognostic targets for IDC.